S(+)-6-methoxy-.alpha.-methyl-2-naphthalene acetic acid of formula (2) belongs to the class of .alpha.-methyl aryl acetic acids also known as 2-aryl propanoic acids which in turn belong to an important class of non-steroidal anti-inflammatory drugs (NSAID). Most commonly used drugs in this class besides naproxen include ibuprofen, ketoprofen and fluriprofen. These drugs have wide applications in checking pain and inflammation caused by arthritis and related connective tissue diseases (Shen, T. Y.; Angew, Chem, Int.Ed., 1972, 11, 460). These drug molecules being chiral in nature appear as racemates, when synthesised through a normal chemical synthetic process. In recent years, the use of enantiomerically pure drugs in chemotherapy is becoming almost mandatory and FDA's of many countries are bringing in new drug legislations for this purpose. The use of enantiomerically pure drugs, not only improves the specificity of action, but also minimises the toxicity and undesirable load on the host. The validity of this statement is very true for .alpha.-aryl propanoic acid also. In case of (.+-.)-6-methoxy-.alpha.-methyl-2-naphthalene acetic acid, the S(+) enantiomer is 28 times more active than its R(-) enantiomer (Roszokwski, A. P., Rooks, W. H., Tomolonis, A. J. and Miller, L. M. J. Pharmcol Exp. Ther, 1971, 179, 114). Another drug belonging to the same class of non-steroidal anti-inflammatory drug (NSAID) which till recently was being prescribed as a racemic mixture is ibuprofen. It has been observed that although the inactive R(-)-antipode of this drug is converted to S(+)-enantiomer in vivo via a CoA thioester intermediate, the epimerisation process leads to metabolic complications as R(-)-ibuprofen-CoA complex competitively inhibits many CoA dependent reactions, which results in perturbation of hepatocyte intermediary metabolism and mitochondrial function. Pure S(+)-ibuprofen may therefore be a preferred drug of future (Ann. Rep. Med. Chem. Vol 30(1995) p.298, Ed. James a Bristol, Academic Press inc. California).
As the chemical synthesis of compound (2) leads to the formation of racemic mixture, one or the other resolution techniques are employed for the separation of S(+) enantiomer (Harrison, I. T., Lewis, B., Nelson, P., Rooks, W., Roszwkoski, A., Tomolonis, A. J. and Fried, J. H. J. Med. Chem., 1970 13 203). These methods generally employ the selective crystallisation of d1-stereoisomeric salts by the use of expensive optically active amines such as cinconidine, dehydroabietyl amine acetate, phenyl ethyl amine etc. (Newman, P. in Optical Resolution Process for Organic Compound Vol.2(II) p.653 (1981), Manhattan College, New York). Resolution of (2) has also been carried out using camphor-10-sulfonic acid (Tsuchihashi, G., Tetrahedron Lett, 1982, 23, 5427).
Asymmetric synthesis of .alpha.-aryl propanoic acids offers another important methodology for obtaining enantiomerically pure compounds. Various strategies employed in these methods include Lewis acid catalyzed 1,2-aryl-migration, use of chiral catalysts in stereoselective C--C bond formation, hydroformylation, asymmetric hydrogenation etc. Asymmetric synthesis of .alpha.-aryl propanoic acids has recently been reviewed [Sonawane, H. R., Bellur, N. S., Ahuja, J. K. and Kulkarni, D. G., Tetrahedron Asym. 1992, 3(2), 162; Vill. C., Giordans, M., Pannosion, S. Sheldrak, G. N. in Naproxen: Industrial Asymmetric syn. (1992). p.303 Ed. Collins, A. N. and Crosby, J. N., Chechester, U.K.: Jia, C., Le, J., Zhonogguo Yiyao Zazhi 1990, 21(3) 137].
A number of reports related to the bio-resolution of (.+-.)-naproxen have appeared in last ten years using hydrolases, lipases, esterases or proteases from bacterial, fungal or animal sources. A brief review is presented in the following lines.
Candida cylinderacea lipase was successfully used for the separation of S(.+-.)-6-methoxy-.alpha.-methyl-2-naphthalene acetic acid through its chloromethyl ester (Gu, Qui-Ming; Chen, C. and Sih, C. J., Tetrahedron Lett. 1986, 27 (16), (1763). This work has also been patented (Sih, C. J., Eur. Pat. Appl. E.P. 227, 078, 01 Jul., 1987, U.S. Appl. 811, 260, 20.sup.th Dec., 1985). Some other important publications in resolution methods for (.+-.)-naproxen and related .alpha.-methyl aryl acetic acids derivatives include (Quax, W. J, Broekhuizen, C. P., Applied Microbial. Biotechnol. 1994, 41 (4), 425; Smeets, J. W. H., Kieboom, A. P. G., Recl. Trav. Chim. Pays-Bass 1992, 111(11), 490, CA, 118: 212077; Mutasaers, J. H., G. M., Kooreman, H. J., Recl. Trav. Chim. Pays-Bas 1991, 110(05) 185, CA, 115-207622t; Gu, Q.; Zhongguo Yiyao Gongyu Zazhi, 1991, 22(21) 49, CA, 115: 88688; Alkumark, S., Anderson, S., Chirality 1992, 4(1) 24, Wu, S. H., Guo, Z. W., Sih, C. J., J. Am. Chem. Soc. 1990, 112(5), 1990) Enantioselective esterification of racemic naproxen has also been carried out using Candida lipase in organic solvents] Shan-Wei, T., Hwa-Jou, W., Biocat., 1994, 11(1), 33 and J. Chem. Technol. Biotechnol., 1996, 65(3), 156].
A few other processes have been patented in the past for the kinetic resolution of (.+-.)naproxen using different enzymes. For example the alkyl esters of (.+-.)-naproxen were claimed to be resolved by the use of enzymes from Pseudomonas, Brevibacterium or Mycoplana species (Watanabe, I., Hosoi, A, Kobayashe, E., J.P., 6363396, 19 Mar., 1988, CA, 109:72056). Gist Brocades, employed Bacillus thai and other micro-organisms to hydrolyse alkyl esters of (.+-.)naproxen (Gist Brocades, N.V., JP 63, 45,234 26 Feb., 1988, FR. Appl. 88.245 7 Jan, 1986, CA, 109: 168975). Resolution of esters of naproxen stereoisomers was achieved in a multiphase extractive membrane bioreactors in presence of Candida cyclinderacea; optically active naproxen was collected in aqueous phase (Matson, S. L. PCT Int. Appl. WO, 88,07,582 06 Oct., 1988 US Appl. 33, 962, 01 Apr., 1987: CA, 111; 113732). A process for the continuous manufacture of S(+)-naproxen was disclosed by Bianchi et al using corresponding alkyl, phenyl, tetrahydropyranyl or tetrahydrofuranyl esters catalyzed by immobilised Candida cylinderacea lipase. They obtained 1757 g of S(+) naproxen from 9387 g of (.+-.)ester after 1200h of continuous operation of the reaction (Bianchi, D., Cesti, P., Pina, C., Battislet, E, Eur. Pat. Appl. E.P. 330, 217, 30 Aug., 1989, IT, 88/19, 532, 25 Feb., 1988, CA 112: 215, 202). Water soluble esters of (.+-.) naproxen were hydrolyzed to produce chiral aryl propinoic acids in a two stage extractive membrane reactor (Matson, S. L., Wald, S. A., Zepp, C. M., and Dodds, D. R. PCT Int. Appl. WO 89,09,765, 19 Oct., 1989, US Appl. 178 735, 07 Apr., 1988, CA, 113: 171683). Hydrolytic resolution of (.+-.)-.alpha.-methyl naphthylacetonitrile derivatives was achieved by Cornynebacterium nitrophilus in 98% ee (Yamamoto, K., Otsubo, K., Oishi, K., Eur. Pat. Appl. E.P. 348, 901, 03 Jan., 1990, JP Appl. 88/156, 911, 27 Jun., 1988, CA 113: 76605). In another approach for the production of S(+)-naproxen a filamentous fungi Cordyceps milioris was employed for isomerisation of R(-)-naproxen to S(+) naproxen (Reid, A. J., Phillips, G. T., Marix, A. F. and De Smet, M. J., Eur. Pat. Appl. E.P. 338, 645,25 Oct., 1989 GB Appl. 88/9, 434,21 April, 1988 CA, 113: 38895). Vinyl, ethyl, methyl esters of (.+-.) naproxen were kinetically resolved using various hydrolases, lipases, esterases etc.; where vinyl ester was claimed to yield maximum resolution (Flling, G., Schlingmann, M., Reinhold, K., Ger, Offen, DE., 3, 919029, 13 Dec., 1990, Appl. 10 Jun., 1989. CA, 114; 245930). Liver enzyme from animals such as rabbit, horse, sheep etc. were also used. (Goswami, A., PCT Int. Appl. WO, 9113163; 05 Sep., 1991, US Appl. 484, 362, 21 Feb., 1990, CA, 115: 254315). Novel Exophiala withansia species was identified to be capable of resolving a-substituted propanoic acid into optically active enantiomers; S(+) naproxen was obtained in 92% ee (De Smet, Jose. M., Eur. Pat. Appl. E.P. 386, 848., 12 Sep., 1990, US Appl. 308, 591, 10 Feb., 1989, CA, 115: 7022). MIS. Syntex Pharmaceuticals Co. patented an ester hydrolase gene from Pseudomonas fluorescens cloned in E. coli for the enantioselective hydrolysis of racemic alkyl esters of naproxen (Chan, H. W., Salazar, F. H., EP 414,247,27 Feb. 1991, US Appl. 398, 102, 24 Aug. 1989, CA, 115, 236 99). Water soluble ethyl sulphate of (+) naproxen was converted into R(-) naproxen using Prozyme 6 (Serine protease of Aspergillus onyzae) in 79.8% yields (Dodds D. R. Zepp, C. M., Rossi, R. F. Eur. Pat. Appl. E.P. 461, 043, 11 Dec. 1991, US appl. 535, 303, 08 June, 1990, CA. 116: 150171). Microbes from Brevibacterium, Bacteridium, Micrococcus, Bacillus etc. were cultivated and used for the resolution of .alpha.-aryl propanoic esters in high ee (Battistel, E., Bianchi, D., Cesti, P., Franzosi, G., Tassinori, R., Spezia, S. Eur. Pat. Appl. E.P. 510, 712, 28 Oct., 1992 15 Apr., 1991/M, 1154. 26 Mar., 1991, CA, 118: 58236). Similarly amides and nitrites were hydrolysed enzymatically to corresponding acids for the manufacture of optically pure naproxen (Ootsubo, K., Yamamoto, K., J.P. 0576, 390, 30 Mar., 1993, Appl. 91/228, 560, 15 Aug., 1991, CA, 119: 93701 and Fallow, R. D. Steiglitz, B., PCT Int. Appl. WO 94, 06, 930, 31 Mar., 1994, US Appl. 948, 185, 21 Sept., 1992, CA, 121: 81125). (.+-.)-Naproxen methyl ester was converted in 35% yields to S(+) naproxen using a panel of micro-organisms most suitable being Zoffeillor latipes (Chan, H. W., Freeman, R., Salazar, H., Beck, S. R., Synder, R. C., Cain, R. O., Roberts, C. R., Felix, H., Phelps, P., Heefner, D. L., PCT Int. Appl. WO 93,23,547, 25 Mar., 1993, US Appl. 883,658, 15 May, 1992, CA, 120: 189868). An enzyme from an organism of the genus Ceracystis was identified and its gene when cloned and expressed in E. coli was used for the production of R(-) naproxen in &gt;95% ee and 26% yields (Julie, W., Hazel, B., Anthony, W. R., PCT Int. Appl. WO 9420, 634, 15 Sep., 1994, GB. Appl. 93/4351, 03 Mar., 1993 CA; 122: 8143).
The number of reviews and patents appearing in the last few years for the preparation of optically pure naproxen from the racemic mixture and over the counter (OTC) status of this drug in USA underline the importance of S(.+-.) naproxen and related compounds as an anti-inflammatory drug of choice. Therefore, production of S(.+-.) naproxen from the racemic mixture enzymatically or by other methods both chemical or catalytic has remained on the top of the priorities for many R&D institutions and pharmaceutical companies world over. The major producers of naproxen till date employ routes involving classical resolution of racemates via diastereoisomeric salt crystallisation.
However, for the last ten years more efforts have been directed towards the development of bio-resolution methods. Biological/enzymatic methods of resolution have the advantage over chemical or other conventional methods for being simple, catalytic, relatively economical and environment friendly. Since enzymes are highly stereo-selective and substrate specific, it therefore requires a specific enzyme for specific bio-conversion. The enzymes available commercially for biotransformation may not display the required selectivity and specificity for the desired bio-conversion and may be of academic interest only. Therefore, the identification, selection and generation of a suitable enzyme is an essential requirement for bio-transformations.